Methionyl-tRNA formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action

Sah, S and Varshney, U (2023) Methionyl-tRNA formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action. In: Microbiology (United Kingdom), 169 (2).

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Abstract

Methionyl-tRNA formyltransferase (Fmt)-mediated formylation of Met-tRNAfMet to fMet-tRNAfMet is crucial for efficient initiation of translation in bacteria and the eukaryotic organelles. Folate dehydrogenase-cyclohydrolase (FolD), a bifunctional enzyme, carries out conversion of 5,10-methylene tetrahydrofolate (5,10-CH2-THF) to 10-formyl-THF (10-CHO-THF), a metabolite utilized by Fmt as a formyl group donor. In this study, using in vivo and in vitro approaches, we show that 10-CHO-DHF may also be utilized by Fmt as an alternative substrate (formyl group donor) to formylate Met-tRNAfMet. Dihydrofolate (DHF) formed as a by-product in the in vitro assay was verified by LC-MS/MS analysis. FolD-deficient mutants and Fmt over-expressing strains were more sensitive to trimethoprim (TMP) than the ∆fmt strain, suggesting that the domino effect of TMP leads to inhibition of protein synthesis and strain growth. Antifolate treatment to Escherichia coli showed a decrease in the reduced folate species (THF, 5,10-CH2-THF, 5-CH3-THF, 5,10-CH+-THF and 5-CHO-THF) and increase in the oxidized folate species (folic acid and DHF). In cells, 10-CHO-DHF and 10-CHO-folic acid were enriched in the stationary phase. This suggests that 10-CHO-DHF is a bioactive metabolite in the folate pathway for generating other folate intermediates and fMet-tRNAfMet.

Item Type:	Journal Article
Publication:	Microbiology (United Kingdom)
Publisher:	Microbiology Society
Additional Information:	The copyright for this article belongs to Microbiology Society.
Keywords:	10-formyldihydrofolate; folic acid; folic acid antagonist; methionyl-tRNA formyltransferase, liquid chromatography; metabolism; tandem mass spectrometry, Chromatography, Liquid; Folic Acid; Folic Acid Antagonists; Tandem Mass Spectrometry
Department/Centre:	Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited:	21 Feb 2023 03:55
Last Modified:	21 Feb 2023 03:55
URI:	https://eprints.iisc.ac.in/id/eprint/80562

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